The present invention relates to recombinant DNA which encodes the BsmI restriction endonuclease (endonuclease) as well as two BsmI methyltransferases (methylases, M1 and M2), and expression of BsmI restriction endonuclease from E. coli cells containing the recombinant DNA.
BsmI restriction endonuclease is found in the strain of Bacillus stearothermophilus NUB36 (New England Biolabs"" strain collection #328). It recognizes double-stranded DNA sequence:
5xe2x80x2 GAATGCNI↓ 3xe2x80x2
3xe2x80x2 CTTACT↑GN 5xe2x80x2 (↓/↑ site of cleavage)
and cleaves downstream of its recognition sequence (N1) on the top strand and also cleaves within the recognition sequence on the bottom strand (between G and C of the 5xe2x80x2 GCATTC 3xe2x80x2 sequence) to generate a 2-base 3xe2x80x2 overhanging ends.
Type II and IIs restriction endonucleases are a class of enzymes that occur naturally in bacteria and in some viruses. When they are purified away from other bacterial proteins, restriction endonucleases can be used in the laboratory to cleave DNA molecules into small fragments for molecular cloning and gene characterization.
Restriction endonucleases act by recognizing and binding to particular sequences of nucleotides (the xe2x80x98recognition sequencexe2x80x99) along the DNA molecule. Once bound, they cleave the molecule within, to one side of, or to both sides of the recognition sequence. Different restriction endonucleases have affinity for different recognition sequences. Over two hundred and eleven restriction endonucleases with unique specificities have been identified among the many hundreds of bacterial species that have been examined to date (Roberts and Macelis, Nucl. Acids Res. 27:312-313, (1999)).
Restriction endonucleases typically are named according to the bacteria from which they are derived. Thus, the species Deinococcus radiophilus for example, produces three different restriction endonucleases, named DraI, DraII and DraIII. These enzymes recognize and cleave the sequences 5xe2x80x2TTT↓AAA3xe2x80x2, 5xe2x80x2PuG↓GNCCPy3xe2x80x2 and 5xe2x80x2CACNNN↓GTG3xe2x80x2 respectively. Escherichia coli RY13, on the other hand, produces only one enzyme, EcoRI, which recognizes the sequence 5xe2x80x2G↓AATTC3xe2x80x2.
A second component of bacterial restriction-modification (R-M) systems are the methyltransferase (methylases). These enzymes are complementary to restriction endonucleases and they provide the means by which bacteria are able to protect their own DNA and distinguish it from foreign, infecting DNA. Modification methylases recognize and bind to the same recognition sequence as the corresponding restriction endonuclease, but instead of cleaving the DNA, they chemically modify one particular nucleotide within the sequence by the addition of a methyl group (C5 methyl cytosine, N4 methyl cytosine, or N6 methyl adenine). Following methylation, the recognition sequence is no longer cleaved by the cognate restriction endonuclease. The DNA of a bacterial cell is always fully modified by the activity of its modification methylase. It is therefore completely insensitive to the presence of the endogenous restriction endonuclease. It is only unmodified, and therefore identifiably foreign DNA, that is sensitive to restriction endonuclease recognition and cleavage.
By means of recombinant DNA technology, it is now possible to clone genes and overproduce the enzymes in large quantities. The key to isolating clones of restriction endonuclease genes is to develop a simple and reliable method to identify such clones within complex genomic DNA libraries, i.e. populations of clones derived by xe2x80x98shotgunxe2x80x99 procedures, when they occur at frequencies as low as 10xe2x88x923 to 10xe2x88x924. Preferably, the method should be selective, such that the unwanted majority of clones are destroyed while the desirable rare clones survive.
A large number of type II restriction-modification systems have been cloned. The first cloning method used bacteriophage infection as a means of identifying or selecting restriction endonuclease clones (EcoRII: Kosykh et al., Mol. Gen. Genet. 178:717-719, (1980); HhaII: Mann et al., Gene 3:97-112, (1978); PstI: Walder et al., Proc. Nat. Acad. Sci. 78:1503-1507, (1981)). Since the presence of restriction-modification systems in bacteria enable them to resist infection by bacteriophage, cells that carry cloned restriction-modification genes can, in principle, be selectively isolated as survivors from genomic DNA libraries that have been exposed to phages. This method has been found, however, to have only limited value. Specifically, it has been found that cloned restriction-modification genes do not always manifest sufficient phage resistance to confer selective survival.
Another cloning approach involves transferring systems initially characterized as plasmid-borne into E. coli cloning plasmids (EcoRV: Bougueleret et al., Nucl. Acids. Res. 12:3659-3676, (1984); PaeR7: Gingeras and Brooks, Proc. Natl. Acad. Sci. USA 80:402-406, (1983); Theriault and Roy, Gene 19:355-359 (1982); PvuII: Blumenthal et al., J. Bacteriol. 164:501-509, (1985); Tsp45I: Wayne et al. Gene 202:83-88, (1997)).
A third approach is to select for active expression of methylase genes (methylase selection) (U.S. Pat. No. 5,200,333 and BsuRI: Kiss et al., Nucl. Acids. Res. 13:6403-6421, (1985)). Since R-M genes are often closely linked together, both genes can often be cloned simultaneously. This selection does not always yield a complete restriction system however, but instead yields only the methylase gene (BspRI: Szomolanyi et al., Gene 10:219-225, (1980); BcnI: Janulaitis et al., Gene 20:197-204 (1982); BsuRI: Kiss and Baldauf, Gene 21:111-119, (1983);
and MspI: Walder et al., J. Biol. Chem. 258:1235-1241, (1983)).
A more recent method, the xe2x80x9cendo-blue methodxe2x80x9d, has been described for direct cloning of restriction endonuclease genes in E. coli based on the indicator strain of E. coli containing the dinD::lacZ fusion (Fomenkov et al., U.S. Pat. No. 5,498,535, (1996); Fomenkov et al., Nucl. Acids Res. 22:2399-2403, (1994)). This method utilizes the E. coli SOS response signals following DNA damages caused by restriction endonucleases or non-specific nucleases. A number of thermostable nuclease genes (TaqI, Tth111I, BsoBI, Tf nuclease) have been cloned by this method (U.S. Pat. No. 5,498,535).
Because purified restriction endonucleases, and to a lesser extent, modification methylases, are useful tools for creating recombinant molecules in the laboratory, there is a commercial incentive to obtain bacterial strains through recombinant DNA techniques that produce large quantities of restriction enzymes. Such overexpression strains should also simplify the task of enzyme purification.
The present invention relates to a method for cloning the BsmI restriction endonuclease gene from Bacillus stearothermophilus NUB36. At first the methylase selection method was used to clone the BsmI methylase gene. A methylase positive clone was derived from a plasmid library containing BsmI genomic DNA. However, no apparent BsmI activity was detected in the cell extract of M+ clone.
The DNA insert in the M+ clone was sequenced by primer walking. The clone was found to contain the entire bsmIM1 gene and a small portion (131 bp) of bsmIM2 gene. To the left side of bsmIM1 and bsmIM2 genes, there was one ORF that showed approximately 30% amino acid sequence identity to a DNA partitioning protein (ParA family). Since restriction endonuclease genes are often located adjacent the methylase gene, it was hypothesized that the BsmI endonuclease gene (bsmIR) is probably located to the right side of BsmIM1 and BsmIM2 genes (FIG. 1). Efforts were made to clone the rest of BsmI M2 gene and the entire bsmIR gene by inverse PCR and PCR. After five rounds of inverse PCR and sequencing of the inverse PCR products, the entire sequence of bsmIM2 gene was obtained. An open reading frame (ORF) of 2031 bp was found downstream of BsmI M2 gene and this ORF was named BsmIR gene (FIGS. 1 and 4). Plasmid pBR-BsmIM1 was only partially resistant to BsmI digestion, while pBR-BsmIM2 was fully resistant to BsmI digestion. Both BsmI M1 and M2 genes were amplified by PCR and cloned into vector pBR322 to generate plasmid pBR-BsmIM1andM2. Both BsmI M1 and M2 genes were under the control of TcR promoter and expressed constitutively in E. coli. The plasmid pBR-BsmIM1andM2 was fully resistant to BsmI digestion, indicating sufficient expression from the TcR promoter.
The bsmIR gene was amplified by PCR and cloned into a low copy number T7 expression vector pACYC-T7ter with compatible ends. The expression vector pACYC-T7ter is derived from pACYC184 and has 5-8 copies per cell. It contains 4 copies of E. coli transcription terminators upstream of the T7 promoter. The transcription terminators are expected to reduce the run-off transcription from cryptic E. coli promoter(s) on the vector. Cell extracts were prepared and assyed for BsmI endonuclease activity. Two isolates (#11 and #33) dislayed full BsmI activity. The recombinant BsmI yield was determined to be 2xc3x97106 units per gram of wet cells (see FIG. 5 for the activity assay). The entire bsmIR gene was sequenced to confirm that #11 carried the wild type bsmIR gene sequence.
Because BsmI endonuclease is a thermostable enzyme, the E. coli cell extract containing BsmI was heated at 65xc2x0 C. and denatured proteins were removed by centrifugation. The soluable proteins were loaded onto a heparin Sepharose column. The proteins were eluted with a salt gradient of 50 mM to 1 M NaCl. BsmI activity was assayed for each fractions. The most active fractions were also analyzed on an SDS-PAGE (FIG. 6). The observed molecular mass of BsmI endonuclease on the SDS-PAGE is 77.9 kDa, in close agreement with the predicted molecular mass of 78.1 kDa.